1. Field of Inventive Subject Matter
The inventive subject matter relates to methods, kits, and compositions for detecting enzyme activity in a biological sample. In particular, the inventive subject matter relates to methods, kits, and compositions for detecting von Willebrand factor degrading enzyme activity in a biological sample.
2. Background
Proteases represent a number of families of proteolytic enzymes that catalytically hydrolyze peptide bonds. Principal groups of proteases include metalloproteases, serine proteases, cysteine proteases and aspartic proteases. Proteases, in particular serine proteases, are involved in a number of physiological processes such as blood coagulation, fertilization, inflammation, hormone production, the immune response and fibrinolysis.
Numerous disease states are caused, and can be characterized, by alterations in the activity of specific proteases and their inhibitors. For example emphysema, arthritis, thrombosis, cancer metastasis and some forms of hemophilia result from the lack of regulation of serine protease activities (see, for example, Textbook of Biochemistry with Clinical Correlations, John Wiley and Sons, Inc. N.Y. (1993)). In case of viral infection, the presence of viral proteases have been identified in infected cells. Such viral proteases include, for example, HIV protease associated with AIDS and NS3 protease associated with Hepatitis C. These viral proteases play a critical role in the virus life cycle.
Measurement of changes in the activity of a specific protease is clinically significant in the treatment and management of an underlying disease state. Proteases, however, are not generally easy to assay. Typical approaches include ELISA, using antibodies that bind the protease, or RIA, using various labeled substrates. Using natural substrates, assays are difficult to perform and expensive. With currently available synthetic substrates the assays are expensive, insensitive and nonselective. In addition, many “indicator” substrates require high quantities of protease which often results, in part, in the self destruction of the protease.
Recent approaches to protease detection rely on a cleavage-induced spectroscopic change in a departing chromogen or fluorogen located in the amino acid position on the carboxyl side of the cleavable peptide bond (see, for example U.S. Pat. Nos. 4,557,862 and 4,648,893). However, many proteases require two to as many as six amino acid residues on either side of the scissile bond for recognition of the protease. Thus, such approaches lack protease specificity.
Fluorogenic indicator compositions have been developed in which a “donor” fluorophore is joined to an “acceptor” chromophore by a short bridge containing a 7 amino acid peptide that is the binding site for an HIV protease and linkers joining the fluorophore and chromophore to the peptide (see, e.g., Wang, et al. (1990) Tetra. Letts. 45: 6493-6496). The signal of the donor fluorophore is quenched by the acceptor chromophore through a process believed to involve resonance energy transfer (RET). Cleavage of the peptide results in separation of the chromophore and fluorophore, removal of the quench, and generation of a subsequent signal measured from the donor fluorophore.
Unfortunately, the design of the bridge between the donor and the acceptor disclosed by Wang, et al. led to relatively inefficient quenching, limiting the sensitivity of the assay. In addition, the chromophore absorbed light in the ultraviolet range, reducing the sensitivity for detection in biological samples which typically contain molecules that absorb strongly in the ultraviolet.
More particularly, several types of assays currently exist for diagnosing VWF abnormalities. In the use of such assays, a VWF antigen must be determined for proper diagnosis. These assays variously include: radioimmunoassay (“RIA”) involving competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody; enzyme immunoassay (“EIA”) and enzyme-linked immunosorbent assay (“ELISA”; see, e.g., U.S. Pat. No. 5,202,264) employing color reaction products of enzyme substrate interaction to measure antigen-antibody reaction; and latex immunoassay (LIA) utilizing antibodies bound at their Fv region to latex particles and presenting a Fab region for interaction with antigens present in blood samples (see, e.g., U.S. Pat. No. 5,585,278).
The foregoing immunoassays are relatively simple and widely used, but suffer several disadvantages. The immunoassays require labeled antibodies, which can be quite expensive and entail intrinsic hazards when radioactive labels are used. In addition, the occurrence of non-specific binding of proteins to antigens, the formation of antibody complexes, and the presence of various types of commonly used solid supports, can each increase background noise and reduce sensitivity, resulting in false-positive determinations (see, e.g., Harlow, E., Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory 1988).
Clearly fluorogenic protease indicators that show a high signal level when cleaved, a low signal level when intact, that show a high degree of protease specificity, and that preferably function in the visible light range, are desirable. The inventive compositions, kits, and methods provide these and other benefits. Applicant has successfully produced a recombinant peptide that can be used as a substrate for enzyme activity assays, utilizing a recombinant polypeptide substrate as disclosed in greater detail herein.